EP0599325B1 - B12 conjugates - Google Patents

Abstract

To prepare cobalamin conjugates of the formula (I> B-CO-Sp-P (I> where B is the radical formed from a cobalamin by elimination of a -CONH2 group, P is the cobalamin coupling partner and Sp is a spacer group, a cobalamin-monocarboxylic acid B-COOH is reacted with a chloroformic ester of the general formula (II) <IMAGE> as condensing agent for coupling, in which R<2> is a straight-chain or branched alkyl radical with 1 to 6 carbon atoms, resulting in an activated cobalamin-monocarboxylic acid of the general formula (III) <IMAGE> which is subsequently used as starting material for linkage to the coupling partner P via the spacer Sp. The result of this is, in particular, a cobalamin conjugate with the general formula (XVII) <IMAGE> in which either P1 is a radical formed from a cobalamin by elimination of a -CONH2 group and P2 is the cobalamin coupling partner, or P2 is a radical formed from a cobalamin by elimination of an H atom and P1 is the cobalamin coupling partner, p and q are identical or different and are integers from 2 to 6, and Y is a single bond or a group of the general formula (XVIII) <IMAGE> where r is an integer from 0 to 3 and s is an integer from 1 to 3.

Description

The invention relates to a new cobalamin conjugate, its production and use in the context of immunoassays, in particular for the determination of vitamin B ₁₂ .

The immunological determination of cobalamins (e.g. vitamin B ₁₂ ) is usually carried out in a competitive test in which the sample's cobalamin and radioactively labeled cobalamin compete for a binding protein for cobalamin (intrinsic factor) bound to a solid phase (RIA test). After separation of the solid from the liquid phase, the amount of cobalamin contained in the sample can be determined in one of the two phases from the amount of radioactive labeling contained therein.

Due to the known disadvantages of RIA tests (e.g. radioactive Pollution, disposal) it is desirable to use the radioactive Labeling by a non-radioactive label, e.g. to be replaced by an enzymatic label.

Known coupling techniques for the production of protein-vitamin B ₁₂ conjugates as immunogens are known (Gershman et al., Archives of Biochemistry and Biophysics 153 (1972), 407; vdWeel et al., Clin.Chim.Acta 56 (1974), 143 ; Endres et al., Clin. Chem. 24/3 (1978), 460; U.S. Patent 3,981,863). The protein-vitamin B ₁₂ conjugates prepared according to the above techniques have already been described as immunogens. However, they are sensitive to hydrolysis and can only be formulated as unstable intermediates. The coupling yield is therefore relatively low and, if at all, very difficult to reproduce. The products obtained by the coupling are also difficult to characterize. In addition, an uncontrolled polymerization of the free enzyme takes place as a side reaction.

EP-A 0 378 204 discloses cobalamin acid hydrazides and Conjugates of these substances with glycoproteins. These conjugates can be made in a simple and reproducible way become. However, the coupling principle can only be used for Glycoproteins are used, the hydroxyl groups on Glycosyl residue after oxidation with periodate for formation a hydrazone group are available.

The object underlying the present invention was thus in taking new cobalamin conjugates at least largely avoiding the disadvantages of the prior art provide that in a simple and reproducible manner can be produced and none on the part of the coupling partner Have limitations.

Furthermore, the coupling of cobalamin to the coupling partner due to the high price of cobalamins in good Yields and take place with an acceptable reaction time.

als Kondensationsmittel zur Kupplung umsetzt, worin R² einen geradkettigen oder verzweigten Alkylrest mit 1 bis 6 Kohlenstoffatomen darstellt, wobei eine aktivierte Cobalamin-Monocarbonsäure der allgemeinen Formel (III)

entsteht, die anschließend als Ausgangsmaterial für die Verknüpfung mit dem Kupplungspartner P über den Spacer Sp dient.This object is achieved by a process for the preparation of cobalamin conjugates of the formula (I) B - CO - Sp - P where B is the residue formed from a cobalamin by splitting off a -CONH ₂ group,
P represents the coupling partner of cobalamin and Sp represents a spacer group, which is characterized in that a cobalamin monocarboxylic acid B-COOH with a chloroformate of the general formula (II)

as a condensing agent for coupling, in which R ^{2 is} a straight-chain or branched alkyl radical having 1 to 6 carbon atoms, an activated cobalamin monocarboxylic acid of the general formula (III)

arises, which then serves as the starting material for the connection with the coupling partner P via the spacer Sp.

It was surprisingly found that the use of a chloroformate with good yields and quickly activated activation of the cobalamin causes while other coupling reagents give unsatisfactory results deliver.

The method according to the invention preferably further comprises the use of the cobalt conjugates produced in immunoassays, especially for Determination of cyanocobalamin.

worin

entweder P₁ einen aus einem Cobalamin durch Abspaltung einer -CONH₂-Gruppe gebildeten Rest und P₂ den Kupplungspartner des Cobalamins darstellt,

oder P₂ einem aus einem Cobalamin durch Abspaltung eines H-Atoms (Amidwasserstoff) gebildeten Rest und P₁ den Kupplungspartner des Cobalamins darstellt, p und q gleich oder verschieden und ganze Zahlen von 2 bis 6 sind, und

Y eine Einfachbindung oder eine Gruppe der allgemeinen Formel (XVIII)

ist, wobei r eine ganze Zahl von 0 bis 3 und s eine ganze Zahl von 1 bis 3 ist.

Another object of the invention is a cobalamin conjugate, which is obtainable by the method according to the invention. The cobalamin conjugate according to the invention preferably has the general formula (XVII)

wherein

either P _{1 represents} a radical formed from a cobalamin by splitting off a -CONH ₂ group and P _{2 represents} the coupling partner of the cobalamin,

or P _{2 represents} a radical formed from a cobalamin by splitting off an H atom (hydrogen amide) and P _{1 represents} the coupling partner of the cobalamin, p and q are identical or different and are integers from 2 to 6, and

Y is a single bond or a group of the general formula (XVIII)

where r is an integer from 0 to 3 and s is an integer from 1 to 3.

The grouping in the general formula (XVII) between P ₁ and P ₂ is thus a preferred spacer Sp for a process for the preparation of cobalamin conjugates of the formula (I).

For compounds of the formula (XVII) it is preferred if p is 2 and q is 2 or 6. Furthermore, it is preferred that when Y is a group of formula (XVIII), r 0 or 2 is. Further, s is preferably 2 when Y is a group of formula (XVIII). P, q, r are also particularly preferred and s is 2 (compounds "a" in Fig. 1) or p, q and s is 2 and r is 0 (compounds "b" in Fig. 1) when Y is a group of formula (XVIII). If Y in formula (XVII) is a single bond, p is particularly preferably equal to 2 and q is 2 or 6, such compounds being represented by Use of cystamine available as a spacer component are.

In compounds of the formula (XVIII), regardless of whether it is P ₁ or P ₂ , the cobalamin is introduced in the form of a B-CO-NH group. The binding partner of the cobalamin is preferably introduced into the conjugate (XVII) in one of the forms binding partner-CONH- or binding partner-NHCO-. However, these two forms are only possible if P _{2 is} the binding partner of cobalamin. If, however, P _{1 is} the binding partner of cobalamin in the case of compounds of the formula (XVII), it can only be introduced as binding partner-CONH-.

wobei X einen Liganden des Kobalts darstellt. Der Ligand X kann beispielsweise H₂O (Vitamin B_12A), OH (Vitamin B_12B), NO (Vitamin B_12C), eine Adenosylgruppe (Coenzym B₁₂) oder ein CN (Vitamin B₁₂) darstellen. Vorzugsweise ist X eine CN-Gruppe, so daß das Cobalamin ein Cyanocobalamin ist. Es ist aber auch möglich, daß der Ligand X fehlt.The cobalamin conjugate according to the invention contains 3 components. The cobalamin component preferably has the general formula (IV)

where X represents a ligand of cobalt. Ligand X can represent, for example, H ₂ O (vitamin B _12A ), OH (vitamin B _12B ), NO (vitamin B _12C ), an adenosyl group (coenzyme B ₁₂ ) or a CN (vitamin B ₁₂ ). Preferably X is a CN group so that the cobalamin is a cyanocobalamin. However, it is also possible that the ligand X is missing.

The cobalamin is bound to the spacer group preferably via one of those designated with (b), (d) or (e) Positions, particularly preferably via one of the with (d) or (e) designated positions and most preferably via the position marked with (d).

The spacer group Sp can be bound to these positions by converting the respective acid amide group -CONH ₂ shown in formula (IV) into the free carboxyl group in a manner known per se by acid saponification and isolation of the free carboxylic acids (cf. Yamada and Hogenkamp, J.Biol Chem. 247 (1972), 6266-6270; Anton et al., J.Amer.Chem.Soc. 102 (1980), 2215). It is preferred to work in such a way that incomplete saponification of the acid amide groups present in the cobalamin molecule is achieved so that only one or a few of the corresponding carboxyl groups are released. The compound in which the free carboxyl group is present at a certain desired location can then be isolated from the mixture thus obtained by methods known per se (for example Inorg.Chim.Acta 162 (1989), 151-155). However, it is also possible to start from a mixture of the cobalamin carboxylic acids, although it is expedient and preferable to start from a single purified carboxylic acid for the preparation of a cobalamin conjugate for use in immunoassays in view of the sensitivity of the determination method.

The spacer group is bound to the cobalamin via an acid amide group, i.e. by linking the activated Carboxyl function of the cobalamin derivative according to formula (III) with an amino function of the spacer.

umsetzt, wobei R¹ eine unverzweigte Alkylengruppe mit 2 bis 20 Kohlenstoffatomen darstellt, die durch ein oder mehrere Heteroatome, insbesondere Sauerstoff- oder Schwefelatome, substituiert sein kann und Y Wasserstoff, eine Schutzgruppe oder eine Gruppe der allgemeinen Formel (VI)

ist, worin R³ eine unverzweigte Alkylengruppe mit 1 bis 6 Kohlenstoffatomen darstellt, und das entstehende Produkt der allgemeinen Formel

mit einem reaktiven Kupplungspartner P umsetzt, um ein Cobalamin-Konjugat der Formel (I) zu erhalten.A preferred embodiment of the preparation of cobalamin conjugates of the formula (I) consists in that the activated cobalamin monocarboxylic acid (III) is reacted with a compound of the general formula (V)

where R ^{1 is} an unbranched alkylene group having 2 to 20 carbon atoms, which can be substituted by one or more heteroatoms, in particular oxygen or sulfur atoms, and Y is hydrogen, a protective group or a group of the general formula (VI)

in which R ^{3 represents} an unbranched alkylene group having 1 to 6 carbon atoms, and the resulting product of the general formula

with a reactive coupling partner P to obtain a cobalamin conjugate of the formula (I).

Y is preferably either hydrogen or a group of general formula (VI). Y can also be a protecting group, e.g. a benzyloxycarbonyl or a tert-butyloxycarbonyl (BOC) group to be known after the reaction after Methods can be split off again.

In a particularly preferred embodiment of the present invention, R ^{1 represents} an unsubstituted (CH ₂ ) _n group, where n is an integer from 1 to 5. N is particularly preferably 2. In a further preferred embodiment, R ^{1 represents} a (C ₂ H ₄ O) _m - (CH ₂ ) ₂ group, where m is an integer from 1 to 5. M is particularly preferably 2. R ³ is preferably a (CH ₂ ) ₂ group.

umgesetzt, worin R¹ wie in Formel (V) definiert ist und R³ eine unverzweigte Alkylengruppe mit 1 bis 6, vorzugsweise 2 Kohlenstoffatomen darstellt. Die Verbindung (Va) kann beispielsweise durch Reaktion eines Diamins H₂NR¹H₂N nach Schutz einer Aminofunktion (z.B. durch eine BOC-Schutzgruppe) und anschließender Reaktion mit N-Succinimidyl-S-acetylthiopropionsäure (SATP) synthetisiert werden. Das entstehende Produkt der allgemeinen Formel (VIII)

wird zur Abspaltung der S-Acetylgruppe mit Hydroxylamin behandelt und das resultierende Produkt wird mit einem Maleimid-funktionalisierten Kupplungspartner der allgemeinen Formel (IX)

umgesetzt, wobei als Endprodukt ein Cobalamin-Konjugat der allgemeinen Formel (X)

entsteht.Depending on the importance of group Y, the intermediate (VII) can then react further in a number of different ways. In the first preferred embodiment, the activated cobalamin monocarboxylic acid (III) with a compound of the general formula (Va)

implemented, wherein R ^{1 is} as defined in formula (V) and R ^{3 represents} an unbranched alkylene group having 1 to 6, preferably 2 carbon atoms. The compound (Va) can be synthesized, for example, by reaction of a diamine H ₂ NR ¹ H ₂ N after protection of an amino function (for example by a BOC protecting group) and subsequent reaction with N-succinimidyl-S-acetylthiopropionic acid (SATP). The resulting product of the general formula (VIII)

is treated with hydroxylamine to split off the S-acetyl group and the resulting product is treated with a maleimide-functionalized coupling partner of the general formula (IX)

implemented, the end product being a cobalamin conjugate of the general formula (X)

arises.

The maleimide-functionalized coupling partner (IX) can for example, a protein that is known per se Activated by reaction with a maleimide derivative (e.g. Yoshitake, Eur. J. Biochem. 101 (1979), 395-399).

wird anschließend mit einer Verbindung der allgemeinen Formel (XII), vorzugsweise mit N-Succinimidyl-S-acetylthiopropionat (R³ = (CH₂)₂)

umgesetzt, wobei R³ gemäß Formel (V) definiert ist. Durch diese Reaktion entsteht ein Produkt der allgemeinen Formel (VIII) (siehe oben), das mit einem Maleimid-funktionalisierten Kupplungspartner der Formel (IX) zu einem Cobalamin-Konjugat der Formel (X) umgesetzt werden kann.In a further embodiment of the method according to the invention, the spacer group can be built up directly on the cobalamin. For this purpose, the activated cobalamin monocarboxylic acid (III) with a non-functionalized diamine of the general formula (Vb) H ₂ N - R ¹ - NH ₂ implemented, wherein R ^{1 is} defined according to formula (V). This reaction is preferably carried out with an excess of the amine so that a molecule of cobalamin reacts with a molecule (Vb). The resulting product (XI)

is then with a compound of general formula (XII), preferably with N-succinimidyl-S-acetylthiopropionate (R ³ = (CH ₂ ) ₂ )

implemented, wherein R ^{3 is} defined according to formula (V). This reaction produces a product of the general formula (VIII) (see above) which can be reacted with a maleimide-functionalized coupling partner of the formula (IX) to give a cobalamin conjugate of the formula (X).

An example of a suitable connection of the general Formula (Vb) is cystamine (2,2'-dithiobis (ethylamine)). The by reaction of the cobalamin monocarboxylic acid (III) with cystamine Intermediate (VII) formed can then on the Disulfide bridge, for example as in Synthesis Commun. (1974), 59, J.Org.Chem. 56: 2648-2650 (1991) or Biochemistry 3 (1964), 480-482, split and to one activated coupling partner, preferably a maleimide functionalized Coupling partners are coupled.

umgesetzt, wobei R¹ wie in Formel (V) definiert ist. Das entstehende Produkt der allgemeinen Formel (XIV)

kann anschließend mit der SH-Funktion eines Thiol-funktionalisierten Kupplungspartners HS ∼ P umgesetzt werden, wobei als Endprodukt ein Cobalamin-Konjugat der allgemeinen Formel (XV)

entsteht.A maleimide-functionalized coupling partner was used in the previously mentioned process variants. However, it is also possible to use a maleimide-functionalized cobalamin derivative. For this purpose, an activated cobalamin monocarboxylic acid (III) with a maleimide derivative of the general formula (XIII)

implemented, wherein R ^{1 is} as defined in formula (V). The resulting product of the general formula (XIV)

can then be reacted with the SH function of a thiol-functionalized coupling partner HS ∼ P, the end product being a cobalamin conjugate of the general formula (XV)

arises.

wobei R¹ und R³ gemäß Formel (V) definiert sind. Ein Beispiel für einen bevorzugten Thiol-funktionalisierten Kupplungspartner ist Biotin-DADOO-(S)ATP (XVII):

In this embodiment of the method according to the invention, the thiol-functionalized coupling partner HS ∼ P preferably has the general formula (XVI)

wherein R ¹ and R ^{3 are} defined according to formula (V). An example of a preferred thiol-functionalized coupling partner is biotin-DADOO- (S) ATP (XVII):

For all the production processes mentioned above, it is particularly preferred if R ^{3 represents} a (CH ₂ -CH ₂ ) group. It is further preferred if R ^{1 represents} a (CH ₂ -CH ₂ ) group or a ((C ₂ H ₄ O) ₂ -CH ₂ -CH ₂ ) group.

The third component of the conjugate according to the invention is the Coupling partner P. The coupling partner P is preferred a detectable molecule, i.e. it contains a component immunologically, by affinity binding (e.g. biotin streptavidin or avidin, protein A-γ immunoglobulin, sugar lectin, Antibody concavalin), enzymatic, by fluorescence, Luminescence or nuclear magnetic resonance is detectable. Preferably the coupling partner is immunological, enzymatic or can be demonstrated by affinity binding.

In a preferred embodiment, the coupling partner P a protein. It can be an enzymatically active one Protein, an immunologically detectable protein (e.g. one Antibody or an antibody fragment) or one by affinity binding act on detectable protein. As enzymatic active protein is preferably alkaline phosphatase, Peroxidase or β-galactosidase, particularly preferably polymerized Peroxidase used. The production of more suitable polymerized enzymes or polypeptides is for example known from EP-A 0 175 560. At least two Enzyme molecules linked and then this polymerized enzyme coupled to an antibody or a fragment thereof. The Polymerization of the enzymes is preferably carried out using a cross-linking reagent that is a bifunctional Medium (e.g. glutardialdehyde). To do this, the two too cross-linking partner chemically in the respective protein structure functional groups contained activated and then coupled with the bifunctional agent.

As a protein that is detectable by affinity binding preferably streptavidin, avidin or a derivative thereof, optionally used in polymerized form.

In another preferred embodiment, the coupling partner an immunological or by affinity binding detectable hapten. Examples of preferred immunological detectable haptens are digoxigenin (or derivatives thereof), Fluorescein, p-nitrophenol, saponin etc. An example of a hapten detectable by affinity binding is biotin or a derivative thereof.

Another object of the present invention is the use of the cobalamin conjugates according to the invention in immunoassays, such as ELISA, in particular for the determination of cyanocobalamin (vitamin B ₁₂ ). The conjugates according to the invention can be used, for example, for determining vitamin B ₁₂ by the method described in DE-OS 39 00 650.

However, it is also possible to use the cobalamin conjugates according to the invention in other methods for determining vitamin B ₁₂ (or other cobalamins) instead of a radioactively labeled cobalamin. Such suitable radioimmunoassays are described, for example, in Clin.Biochem. 18 (1985), 261-266, U.S. Patent 3,981,863, Clin.Chim.Acta 56 (1974), 143-149, Lit.Clin.Path. 20 (1967), 683-686, British J. Them. 22 (1972), 21-31.

The following examples and figures 1 to 3 explain the invention further.

Figure 1 shows the chemical structural formulas for the in Examples 1 to 11 prepared compounds (4a), (4b), (6a), (6b), (8a), (8b), (3a), (3b) and (9).

Figure 2 shows the chemical structure of the in the examples 12 to 14 compounds (14), (13) and (15).

Figure 3 shows calibration curves for the B ₁₂ determination, which were obtained using a conjugate according to the invention and a conjugate of the prior art.

Example 1: B _12th -d-DADOO- (S) ATP (4a)

Ausbeute:

148 mg (25 %) B₁₂-d-DADOO-(S)ATP

500 mg (0.37 mmol) of B ₁₂ d acid are dissolved in 30 ml of absolute DMF and 5 ml of DMSO, and 155 μl (1.09 mmol) of triethylamine are added. After stirring at 20 ° C. for 20 min, the reaction mixture is cooled to 0 ° C. and 143 μl (1.09 mmol) isobutyl chloroformate are added. After a further 15 minutes with ice cooling, a solution of 645.4 mg (1.4 mmol) of DADOO- (S) ATP x trifluoroacetic acid (see Example 8), 193 μl (1.4 mmol) of triethylamine and 10 ml of absolute DMF is added. A slight gas development can be observed. The mixture is stirred for a further 30 min at 0 ° C., then the reaction mixture is brought slowly to 20 ° C., the conversion is checked by means of HPLC and the solvent is evaporated off under a high vacuum (HV) (bath temperature not above 50 ° C.). The residue is dissolved in 25 ml of water and desalted on an Amberlite®XAD 2 column (3.5 x 30 cm). After elution of the product with methanol, the solvent is removed in a water jet vacuum (WSV), the residue is dissolved in 25 ml of water and this solution is applied to a Dowex®1x2 column (acetate form, 3.0 x 30 cm). The product is eluted with water and then purified by preparative HPLC. The clean fractions are pooled and lyophilized.

Yield:

148 mg (25%) B ₁₂ -d-DADOO- (S) ATP

Analytical HPLC:

Pillar:

Vydac®C18 MZ-Micro, 300 Å, 5 µm, 4.6 x 250 mm

Eluent:

A: 0.01 m NH ₄ Ac pH 6.7 B: 95% acetonitrile 0.01 m NH ₄ Ac pH 6.7

Gradient:

0 → 80% B in 60 min

Flow:

1 ml / min

Detector:

UV detector, wavelength 226 nm

Preparative HPLC:

Pillar:

Waters Delta-PAK ™ C18, 100 Å, 15 µm, 50 x 300 mm

Eluent:

A: 0.01 NH ₄ Ac pH 6.7 B: 95% acetonitrile 0.01 mol / l NH ₄ Ac pH 6.7

Gradient:

0% B 10 min 0 → 40% B in 110 min 40 → 80% B in 10 min 80% B 20 min

Flow:

15 ml / min

Detector:

UV detector, wavelength 226 nm

Analytical data:

Retention time analytical HPLC:

22.3 min

IR (KBr):

γ = 3350, 3170, 2120, 1660, 1570, 1495, 1398 cm ^-1

¹ H-NMR (D ₂ O, TMS-Na salt):

contains the signals of the B ₁₂ -d-carboxylic acid and 2.30 ppm (s, 3H, C H ₃ -CO); 2:53 (t, 2H, C H ₂ -CO); 3:08 (t, 2H, C H ₂ S); 3:32 (t, 4H, C H ₂ NHCO); 3.54 ppm (m, 8H)

FAB-MS:

1617.5 [M + H] ⁺

Example 2: B _12th -e-DADOO- (S) ATP (4a)

Ausbeute:

62 mg (11 %) B₁₂-e-DADOO-(S)ATP

Implementation analogous to B ₁₂ -d-DADOO- (S) ATP only with B ₁₂ -e-COOH.

Yield:

62 mg (11%) B ₁₂ -e-DADOO- (S) ATP

Analytical data:

Retention time analytical HPLC:

22.6 min

¹ H-NMR (D ₂ O, TMS-Na salt):

contains the signals of the B ₁₂ -e carboxylic acid and 2.33 (s, 3H, C H ₃ -CO); 2.65 (t, 2H, C H ₂ CO); 3.10 (t, 2H, C H ₂ S); 3:39 (t, 4H, C H ₂ NHCO); 3.70 ppm (m, 8H)

³¹ P NMR (D ₂ O, TMS Na salt):

1.6 ppm (s)

FAB-MS:

1617 [M + H] ⁺

Example 3: B _12th -d-ethylenediamine- (S) ATP (4b)

Ausbeute:

60 mg (11 %) B₁₂-d-ED-(S)ATP

500 mg (0.37 mmol) of B ₁₂ d acid are dissolved in 30 ml of absolute DMF and 5 ml of DMSO, and 155 μl (1.09 mmol) of triethylamine are added. After stirring at 20 ° C. for 20 min, the reaction mixture is cooled to 0 ° C. and 143 μl (1.09 mmol) isobutyl chloroformate are added. After a further 15 minutes with ice cooling, a solution of 425.6 mg (1.4 mmol) of ethylenediamine (S) ATP x trifluoroacetic acid, 193 μl (1.4 mmol) of triethylamine and 10 ml of absolute DMF is added. A slight gas development can be observed. Further processing is carried out analogously to that of the B ₁₂ - DADOO- (S) ATP.

Yield:

60 mg (11%) B ₁₂ -d-ED- (S) ATP

Analytical data:

Retention time analytical HPLC:

19.0 min

¹ H-NMR (D ₂ O, TMS-Na salt):

contains the signals of the B ₁₂ -d-carboxylic acid and 2.30 (s, 3H, C H ₃ CO); 2:45 (t, 2H, C H ₂ CO); 2.60 (m, 2H, C H ₂ S); 3:05 (t, 2H, C H ₂ NH); 3.15 ppm (m, 2H, C H ₂ NH)

FAB-MS:

1528.6 [M + H] ⁺

Example 4: N (tert-butoxycarbonyl) -1,8-diamino-3,6-dioxaoctane (6a) Intermediate

Ausbeute:

58.2 g (23.5 % d. Th.)

¹H-NMR (CDCl₃/TMS):

1.45 (s, 9H, O-C(CH ₃)₃); 2.88 (t, 2H, J=5.3 Hz, CH ₂NH₂); 3.28 (t, 2H, J=5.7 Hz, CH ₂NH); 3.52 (m, 4H, CH ₂O); 3.57 (s, 4H, CH ₂O); 5.21 ppm (s, br, 1H, NHCO)

DC:

Kieselgel 60 (Merck),
Isopropanol/Butylacetat/Wasser/Ammoniak (v/v/v/v 50/30/15/5),
Detektion mit Ninhydrin.
R_f = 0.45

150 g (1 mol) of 1,8-diamino-3,6-dioxaoctane (DADOO) in 900 ml of dioxane / water (v / v 1/1) are slowly stirred with a solution of 109 g (0.5 mol) of di -tert.-butyl dicarbonate in 450 ml of dioxane. After the dropwise addition, the reaction mixture is stirred for a further 1.5 h at 20 ° C., then the solvent is distilled off and the residue is taken up in 1000 ml of ethyl acetate / water (v / v 1/1). After the aqueous phase has been separated off, the organic phase is extracted twice with 500 ml of 0.1N hydrochloric acid each. The aqueous phases are combined, the pH is adjusted to pH 9-10 with dilute sodium hydroxide solution and the solution is subjected to a liquid-liquid extraction in the perforator. After 8 hours of extraction with 750 ml of ethyl acetate, the solvent is removed and the residue is dried under HV.

Yield:

58.2 g (23.5% of theory)

¹ H-NMR (CDCl ₃ / TMS):

1:45 (s, 9H, OC (C H _{3) 3);} 2.88 (t, 2H, J = 5.3 Hz, C H ₂ NH _2); 3.28 (t, 2H, J = 5.7 Hz, C H ₂ NH); 3:52 (m, 4H, C H ₂ O); 3:57 (s, 4H, C H ₂ O); 5.21 ppm (s, br, 1H, N H CO)

DC:

Kieselgel 60 (Merck),
Isopropanol / butyl acetate / water / ammonia (v / v / v / v 50/30/15/5),
Detection with ninhydrin.
R _f = 0.45

Example 5: 1-butoxycarbonyl-1,2-diaminoethane (m-Boc-ED) (6b) Intermediate

Ausbeute:

27.3 g (8.5 % d. Th.)

¹H-NMR (CDCl₃/TMS):

1.45 (s, 9H, O-C(CH ₃)₃); 2.79 (T, 2H, J=7.5 Hz, CH ₂-NH); 3.14 (m, 4H, CH ₂-NH₂); 5.00 ppm (s, br, 1H, O-CONH)

DC:

Kieselgel 60 (Merck), Isopropanol/Butylacetat/Wasser/Ammoniak (v/v/v/v/ 50/30/15/5), Detektion mit Ninhydrin.
R_f = 0.23

218 g (1 mol) of di-tert-butyl diarbonate in 1 l of dioxane are added to a solution of 120 g (2 mol) of ethylenediamine and 2 l of dioxane / water (v / v 1/1) with stirring at 0 ° C. within 1 h dripped. After stirring for one hour at 20 ° C., the solvent is concentrated in a water jet vacuum, the bis-1,2-butoxycarbonyl-1,2-diaminoethane formed as a by-product is filtered off and the filtrate is extracted with 300 ml of ethyl acetate. The organic phase is washed three more times with 100 ml of water each time, dried over sodium sulfate and concentrated in a water jet vacuum.

Yield:

27.3 g (8.5% of theory)

¹ H-NMR (CDCl ₃ / TMS):

1:45 (s, 9H, OC (C H _{3) 3);} 2.79 (T, 2H, J = 7.5 Hz, C H ₂ -NH); 3.14 (m, 4H, C H ₂ -NH _2); 5.00 ppm (s, br, 1H, O-CON H )

DC:

Kieselgel 60 (Merck), isopropanol / butyl acetate / water / ammonia (v / v / v / v / 50/30/15/5), detection with ninhydrin.
R _f = 0.23

Example 6: 1-butoxycarbonyl-8 - [(3-methylcarbonylthio) propionyl] -1,8-diamino-3,6-dioxaoctane (Boc-DADOO- (S) ATP) (8a)

Ausbeute:

11.97 g ( 63.2 % d. Th.)

¹H-NMR (CDCl₃/TMS):

1.45 (s, 9H, O-C(CH ₃)₃); 2.33 (s, 3H, CH ₃); 2.51 (t, 2H, J=7.0 Hz, CH ₂CO); 3.14 (t, 2H, J=7.0 Hz,CH ₂S); 3.56 (m, 12H); 5.39 (s br, 1H, NH-COO); 6.79 ppm (s br, 1H, NH-CO)

IR (Film):

3300, 2980, 1740, 1680, 1520 cm^-1

12.41 g (50 mmol) of 6a and 12.26 g (50 mmol) of N-succinimidyl-S-acetylthiopropionate (SATP) are dissolved in 100 ml of tetrahydrofuran and stirred at 20 ° C. for 4 h. The solvent is then distilled off, the residue is taken up in 250 ml of ethyl acetate / water (v / v 1/1) and adjusted to pH 4-5 with 6N HCl. The organic phase is separated off, extracted twice with 100 ml of water each time, dried over sodium sulfate, the solvent is distilled off and the residue is dried under high vacuum.

Yield:

11.97 g (63.2% of theory)

¹ H-NMR (CDCl ₃ / TMS):

1:45 (s, 9H, OC (C H _{3) 3);} 2:33 (s, 3H, C H _3); 2.51 (t, 2H, J = 7.0 Hz, C H ₂ CO); 3.14 (t, 2H, J = 7.0 Hz, C H ₂ S); 3.56 (m, 12H); 5.39 (s br, 1H, N H -COO); 6.79 ppm (s br, 1H, N H -CO)

IR (film):

3300, 2980, 1740, 1680, 1520 cm ^-1

Example 7: 1-butoxycarbonyl-2 - [(3-methylcarbonylthio) propionyl] -1,2-diamino-ethane (Boc-ED- (S) ATP) (8b)

Ausbeute:

9.22 g (63.9 % d. Th.)

¹H-NMR (CDCl₃/TMS):

1.44 (s, 9H, O-C(CH ₃)₃); 2.32 (s, 3H, - CH ₃); 2.52 (t, 2H, J=7.0 Hz, CH ₂CO); 3.14 (t, 2H, J=7.0 Hz, CH ₂S); 3.33 (m, 4H, CH ₂-NH); 5.00 (s, br, 1H, OCONH); 6.37 ppm (s, br, 1H, CONH)

DC:

Kieselgel 60 (Merck), Isopropanol/Butylacetat/Wasser/Ammoniak (v/v/v/v 50/30/15/5), Detektion mit Ninhydrin.
R_f = 0.87

8.00 g (50 mmol) of 6b and 12.26 g (50 mmol) of N-succinimidyl-S-acetylthiopropionate (SATP) are dissolved in 100 ml of tetrahydrofuran and stirred at 20 ° C. for 4 h. The solution is then cooled in an ice bath, the precipitated product is suctioned off and dried in a high vacuum at 40 ° C.

Yield:

9.22 g (63.9% of theory)

¹ H-NMR (CDCl ₃ / TMS):

1:44 (s, 9H, OC (C H _{3) 3);} 2:32 (s, 3H, - C H _3); 2.52 (t, 2H, J = 7.0 Hz, C H ₂ CO); 3.14 (t, 2H, J = 7.0 Hz, C H ₂ S); 3:33 (m, 4H, C H ₂ -NH); 5.00 (s, br, 1H, OCON H ); 6.37 ppm (s, br, 1H, CON H )

DC:

Kieselgel 60 (Merck), isopropanol / butyl acetate / water / ammonia (v / v / v / v 50/30/15/5), detection with ninhydrin.
R _f = 0.87

Example 8: 1 - [(3-Methylcarbonylthio) propionyl] -1,8-diamino-3,6-dioxaoctane trifluoroacetate (DADOO- (S) ATP x CF _3rd COOH) (3a)

Ausbeute:

9.41 g (100 % d. Th.)

¹H-NMR (CDCl₃/TMS):

2.30 (s, 3H, CH ₃); 2.52 (t, 2H, J=7.0 Hz, CH ₂-CO); 3.08 (t, 2H, J=7.0 Hz, CH ₂-S); 3.62 (m, 12H); 7.05 (s br, 1H, NH-CO); 7.64 ppm (s br, 3H, NH)

DC:

Kieselgel 60 (Merck), Essigester/Eisessig/Wasser (v/v/v 5/5/2), Detektion mit Ninhydrin.
R_f = 0.58

9.46 g (25 mmol) 8a are dissolved in 50 ml trifluoroacetic acid and stirred at 0 ° C. After the evolution of gas has ended, the reaction mixture is slowly brought to 20 ° C. and the solvent is distilled off in a water jet vacuum. The oily residue is dried in a high vacuum.

Yield:

9.41 g (100% of theory)

¹ H-NMR (CDCl ₃ / TMS):

2.30 (s, 3H, C H _3); 2.52 (t, 2H, J = 7.0 Hz, C H ₂ -CO); 3.08 (t, 2H, J = 7.0 Hz, C H ₂ -S); 3.62 (m, 12H); 7.05 (s br, 1H, N H -CO); 7.64 ppm (s br, 3H, N H )

DC:

Kieselgel 60 (Merck), ethyl acetate / glacial acetic acid / water (v / v / v 5/5/2), detection with ninhydrin.
R _f = 0.58

Example 9: 1 - [(3-Methylcarbonylthio) propionyl] -1,2-diaminoethane trifluoroacetate (ED- (S) ATP x TFA) (3b)

Ausbeute:

5.54 g (65 % d. Th.)

¹H-NMR (D₆-DMSO/TMS):

2.31 (S, 3H, -CH ₃); 2.39 (t, 2H, J=7.0 Hz, CH ₂-CO); 2.85 (t, 2H, J=7.0 Hz, CH ₂S); 3.00 (t, 2H, J=7.0 Hz, CH ₂NH); 3.26 (t,2H, J=7.0 Hz, CH ₂NH); 7.92 (s, br, NH); 8.13 ppm (s, br, 1H, NH-CO)

DC:

Kieselgel 60 (Merck), Essigester/Eisessig/Wasser (v/v/v 5/5/2), Detektion mit Ninhydrin.
R_f = 0.62

7.25 g (25 mmol) 8b are dissolved in 10 ml trifluoroacetic acid and stirred at 20 ° C for 4 h. The solution is then evaporated to dryness in a water jet vacuum and the residue is digested with 50 ml of diisopropyl ether. The product is dried in a high vacuum and crystallizes out after a longer induction phase.

Yield:

5.54 g (65% of theory)

¹ H-NMR (D ₆ -DMSO / TMS):

2.31 (S, 3H, -C H ₃ ); 2.39 (t, 2H, J = 7.0 Hz, C H ₂ -CO); 2.85 (t, 2H, J = 7.0 Hz, C H ₂ S); 3:00 (t, 2H, J = 7.0 Hz, C H ₂ NH); 3.26 (t, 2H, J = 7.0 Hz, C H ₂ NH); 7.92 (s, br, N H ); 8.13 ppm (s, br, 1H, N H -CO)

DC:

Kieselgel 60 (Merck), ethyl acetate / glacial acetic acid / water (v / v / v 5/5/2), detection with ninhydrin.
R _f = 0.62

Example 10: B ₁₂ -d-DADOO and B ₁₂ -e-DADOO (9)

Ausbeute:

49 mg (9 %) B₁₂-d-DADOO
160 mg (29 %) B₁₂-e-DADOO

500 mg (0.37 mmol) of the corresponding B ₁₂ monocarboxylic acid are dissolved in 30 ml of absolute DMF and 5 ml of DMSO, and 155 μl (1.09 mmol) of triethylamine are added. After stirring at 20 ° C. for 20 min, the reaction mixture is cooled to 0 ° C. and 143 μl (1.09 mmol) isobutyl chloroformate are added. After a further 15 minutes with ice cooling, 548 mg (3.7 mmol) of diaminodioxaoctane are added. A slight gas development can be observed. The further procedure is analogous to the implementation with DADOO- (S) ATP.

Yield:

49 mg (9%) B ₁₂ -d-DADOO
160 mg (29%) B ₁₂ -e-DADOO

Analytical data B _12th -d-DADOO:

Retention time of analytical HPLC:

19.5 min

IR (KBr):

γ = 3350, 3170, 2120, 1660, 1570, 1495, 1398 cm ^-1

¹ H-NMR (D ₂ O, TMS-Na salt):

contains the signals of the B ₁₂ -d-carboxylic acid as well as 3.20 (t, 4H), 3.70 ppm (m, 8H)

FAB-MS:

1486.6 [M] ⁺

Analytical data B _12th -e-DADOO:

Retention time of analytical HPLC:

19.5 min

¹ H-NMR (D ₂ O, TMS-Na salt):

contains the signals of the B ₁₂ -e carboxylic acid and 3.22 (2t, 4H), 3.73 ppm (m, 8H)

³¹ P NMR (D ₂ O, TMS Na salt):

1.48 (s)

FAB-MS:

1486.9 [M + H] ⁺

Example 11: B _12th -d-DADOO- (S) ATP (4a)

5 mg (3.7 x 10 ^-3 mmol) of B ₁₂ -d-DADOO ( 9 ) are dissolved in 1 ml of DMF, a spatula tip of SATP, also dissolved in 1 ml of DMF, is added and the mixture is stirred at 20 ° C. for 5 h. The purification takes place by means of HPLC.

Analytical data:

Retention time:

22.3 min

Example 12: Biotin-DADOO- (S) ATP (14)

400 mg (1.07 mmol) of Biotin-DADOO are dissolved in 10 ml of DMF, mixed with 262 mg (1.07 mmol) of SATP and stirred at 4 ° C. for 4 d. After removing the solvent, the residue is digested with ethyl acetate, suction filtered and dried. The crude product is purified by means of open column chromatography (SiO ₂ , methanol / ethyl acetate (v / v 1/1).

Analytical data:

Retention time:

Biotin-DADOO- (S) ATP: 22.10 min

¹ H-NMR (D ₆ -DMSO, TMS):

1:40 (m, 6H, 3 C H _2); 2:06 (t, 2H, C H ₂ CO); 2.31 (s, 3H, C H ₃ CO); 2:37 (t, 2H, C H ₂ CO); 2.77 (d, 1H, C H S); 2.99 (t, 2H, C H ₂ S); 3.16-3.70 (m, 14H); 4.22 (m, 2H, 2 C H NH); 6.40 (d, 2H, 2NH ); 7.85 (t, 1H, N H CH _2); 8.01 ppm (t, 1H, N H CH ₂ )

Example 13: B ₁₂ -d-MEA (13)

50 mg (0.037 mmol) of B ₁₂ -d acid are dissolved in 3 ml of DMF and 2 ml of DMSO, and 16 μl (0.109 mmol) of triethylamine are added. After stirring for 15 minutes at 20 ° C., the mixture is cooled to 0 ° C., 14 μl (0.109 mmol) isobutyl chloroformate are added and the mixture is stirred at 0 ° C. for a further 20 minutes. A solution of 19.5 mg (0.111 mmol) of maleimidoethylamine hydrochloride (MEA x HCl) in 3 ml of DMF / triethylamine 2/1 (v / v) is then added dropwise and the mixture is stirred at 20 ° C. for 1 h. The crude product is subjected to the subsequent reaction without further purification.

Analytical data:

Retention times:

MEA x HCl: 9.81 min
B ₁₂ -d-COOH: 11.80 min
B ₁₂ -d-MEA: 20.33 min

Example 14: B _12th -d-MEA- (SA) TP-DADOO-biotin (15)

30 mg (0.059 mmol) of biotin-DADOO- (S) ATP 2 are dissolved in 5 ml of 0.025 M hydroxylamine solution and stirred at 20 ° C. for 1 h under an argon atmosphere. The solvent is concentrated in a water jet vacuum and the residue is taken up in 2 ml of DMF. This solution is combined with the reaction solution 13 and stirred at 20 ° C. for 1 h. After the solvent has been separated off and desalted using Amberlite®XAD 2, the product is purified using preparative HPLC.

Analytical data:

Retention time:

Biotin-DADOO- (SA) TP: 19.95 min
B ₁₂ -d-MEA- (SA) TP-DADOO-biotin: 23.05 min

Example 15: Conjugation of B ₁₂ -d-DADOO- (S) ATP to prepolymerized peroxidase (pPOD)

Prepolymerized POD can, for example, with glutardialdehyde, as described by Engvall and Perlmann
(Immunochemistry 8 (1971), 871-874).

a) Activation of pPOD with maleimidohexanoyl-N-hydroxysuccinimide ester (MHS)

Maleimide groups are introduced into pPOD according to Yoshitake, Eur.J.Biochem. 101 , 395-399 (1979):
MHS is freshly dissolved in DMSO immediately before use (160 mg / ml DMSO). pPOD is adjusted to a protein concentration of 25 mg / ml in 0.04 mol / l potassium phosphate buffer pH 7.1. 30 µl of the MHS solution are pipetted into 1 ml of the pPOD solution, so that a molar POD: MHS ratio of 1:25 is achieved. After incubating the resulting mixture at 25 ° C. for 1 hour, 10 μl of a lysine solution (1 mol / l) are pipetted into each ml of reaction mixture and incubated at 25 ° C. for a further 30 minutes. The activated pPOD solution (pPOD-MH) is dialyzed overnight with cooling against a 1000-fold volume of pre-cooled buffer (100 mmol / l potassium phosphate / 1 mmol / l EDTA / pH 6.0). After dialysis, pPOD-MH is adjusted to a protein concentration of 10 mg / ml.

b) Activation of B ₁₂ -d-DADOO- (S) ATP

20 mg B ₁₂ -d-DADOO- (S) ATP are incubated in 4 ml H ₂ O with 40 µl hydroxylamine solution (2 mmol / l pH 6.3) at 25 ° C for 20 minutes.

c) Conjugation of B ₁₂ -d-DADOO- (S) ATP to pPOD-MH

5 ml of the pPOD-MH solution (50 mg pPOD-MH) are incubated with 400 μl of the activated B ₁₂ -d-DADOO- (S) ATP solution with stirring at 25 ° C. for 3 hours. To stop the reaction, 10 μl of a cysteine solution (1 mol / l in H ₂ O) are pipetted in, so that a cysteine concentration of 2 mmol / l results. After a further 30 minute incubation at 25 ° C., 270 μl of a 100 mmol / l N-methylmaleinimide solution (NMM) in DMSO (11 mg / ml) are pipetted in, and an NMM concentration of 5 mmol / l is set and another 60 Incubated for minutes at 25 ° C.

The conjugation approach is used for purification using a Sephadex®G-25 chromatographed coarse column. As a running buffer 40 mmol / l potassium phosphate buffer, pH 6.0 used.

The purified B ₁₂ -d-DADOO-S-pPOD conjugate is concentrated to a peroxidase activity of approx. 2000 U / ml.

Example 16: Determination of vitamin B ₁₂

The determination of vitamin B ₁₂ is carried out analogously to the description in EP-A 0 378 197.

a) Sample preparation

The sample preparation, ie the detachment of the B ₁₂ from its binding protein, can be carried out analogously to EP-A 0 378 204 by treatment with lipoic acid or by exposure to heat or by destroying the binding protein in the alkaline range (pH> 13.5).

250 µl of human serum are mixed with 125 µl of detachment reagent (consisting of from 8 mg / ml lipoic acid, 1 mg / ml potassium cyanide dissolved in 0.5 mol / l NaOH) mixed and incubated for 15 minutes at room temperature. Then 125 ul of a phosphate buffer solution (200 mmol / l; pH 4.1) added.

b) reagents

Polystyrene tube coated with Thermo-RSA streptavidin (manufactured according to EP-A 0 269 092)

Reagent 1

MAK-BI Konzentration in Verbindung mit Reagenz 2a: 6 ng/ml

MAK-BI Konzentration in Verbindung mit Reagenz 2b: 75 ng/ml

Biotinylated monoclonal antibody (MAK-BI) from EP-A 0 378 197, biotinylated according to JACS 100 (1978), 3585-3590; 40 mmol / l phosphate buffer, pH 7.2.

MAK-BI concentration in combination with reagent 2a: 6 ng / ml

MAK-BI concentration in combination with reagent 2b: 75 ng / ml

Reagent 2a B ₁₂ -d-DADOO-S-pPOD

20 mmol/l Na-Phosphatpuffer;

10 mmol/l EDTA; 150 mmol/l NaCl; pH 6,8

0,25 % Rinderserumalbumin (RSA); 0,05 % Tween® 20

Activity: 500 mU / ml

20 mmol / l Na phosphate buffer;

10 mmol / l EDTA; 150 mmol / l NaCl; pH 6.8

0.25% bovine serum albumin (RSA); 0.05% Tween® 20

Reagent 2b (comparative conjugate according to EP-A 0 378 203) B ₁₂ -d-CO-NH-NH-CO-CH ₂ - (- O-CH ₂ -CH ₂ -) ₃ -O-CH ₂ -CO-NH-N = POD (= B ₁₂ -TEDEH-POD)

20 mmol/l Na-Phosphatpuffer;

10 mmol/l EDTA; 150 mmol/l NaCl; pH 6,8

0,25 % RSA, 0,05 % Tween® 20

Activity: 30 mU / ml.

20 mmol / l Na phosphate buffer;

10 mmol / l EDTA; 150 mmol / l NaCl; pH 6.8

0.25% RSA, 0.05% Tween® 20

Reagent 3

100 mmol / l phosphate citrate buffer, pH 4.4.

1.9 mmol / l ABTSs

3.2 mmol / l sodium perborate.

c) implementation

To carry out the determination, 100 μl of pretreated sample or standard solution with 500 μl of reagent 1 (MAK solution) are pipetted into a streptavidin tube and incubated at 25 ° C. for 30 minutes. 500 μl of reagent 2 (conjugate solution) are then pipetted in and incubated at 25 ° C. for a further 60 minutes. It is washed with washing solution and 1000 μl of reagent 3 (substrate solution) are added, incubated for 30 minutes at 25 ° C. and the color formed is measured as a measure of vitamin B ₁₂ content at 422 nm.

Fig. 3 shows the comparison of B ₁₂ calibration curves obtained with the conjugate according to the invention (reagent 2a, curve 1) and a conjugate of the prior art (reagent 2b, curve 2).

Thereafter it turns out that with the conjugate according to the invention steeper and thus better calibration curves can be achieved.

Claims (12)

1. Process for the production of cobalamin conjugates of formula (I) B - CO - Sp - P in which B represents the residue formed from a cobalamin by cleavage of a -CONH₂ residue,
   P represents a detectable molecule as coupling partner of the cobalamin and Sp represents a spacer group,
   wherein
   a cobalamin monocarboxylic acid B-COOH is reacted with a chloroformic acid ester of the general formula (II)

   

   as the condensing agent for the coupling, in which R² represents a straight-chained or branched alkyl residue with 1 to 6 carbon atoms, thereby forming an activated cobalamin monocarboxylic acid of the general formula (III)

   

   which subsequently serves as the starting material for linkage to the coupling partner P via the spacer Sp.
2. Process as claimed in claim 1,
   wherein
   the cobalamin has the general formula (IV)

   

   in which X represents a ligand of cobalt and the cobalamin is linked to the spacer group via one of the positions denoted (b), (d) or (e).
3. Process as claimed in claim 1 or 2,
   wherein
   the activated cobalamin monocarboxylic acid of the general formula (III) is reacted with a maleimide derivative of the general formula (XIII)

   

   in which R¹ represents an unbranched alkylene group with 2 to 20 carbon atoms which can be substituted by one or several heteroatoms, in particular by oxygen or sulphur atoms, the product which forms of the general formula (XIV)

   

   is reacted with the SH group of a coupling partner HS ∼ P provided with a thiol functional group to form a cobalamin conjugate of the general formula

   
4. Cobalamin conjugate having the general formula (XVII)

   

   in which

   P₁ either represents a residue formed from a cobalamin by cleavage of a -CONH₂ residue and P₂ represents a detectable molecule as the coupling partner of the cobalamin or P₂ represents a residue formed from a cobalamin by cleavage of a H atom and P₁ represents a detectable molecule as the coupling partner of the cobalamin,

   p and q are the same or different and are integers from 2 to 6 and

   Y is a single bond or a group of the general formula (XVIII)

   

   in which r is an integer from 0 to 3 and s is an integer from 1 to 3.
5. Conjugate as claimed in claim 4,
   wherein
   the cobalamin has the general formula (IV) according to claim 2.
6. Conjugate as claimed in claim 5,
   wherein
   binding takes place via one of the positions denoted (d) or (e).
7. Conjugate as claimed in claim 5 or 6,
   wherein
   X is a cyano group.
8. Conjugate as claimed in one of the claims 5 to 7,
   wherein
   the coupling partner of the cobalamin is a peroxidase, β-galactosidase, alkaline phosphatase or streptavidin.
9. Conjugate as claimed in one of the claims 5 to 7,
   wherein
   the coupling partner of the cobalamin is biotin.
10. Use of cobalamin conjugates as claimed in one of the claims 4 to 9 in immunological tests, in particular for the determination of cyanocobalamin.
11. Use as claimed in claim 10 in an ELISA test.
12. Process as claimed in one of the claims 1 to 3 additionally comprising the use of the produced cobalamin conjugates in immunological tests, in particular for the determination of cyanocobalamin.

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